Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are not adequately specific, and the medical profession has come to rely on serum biochemical markers of cardiac tissue necrosis for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used, and subsequently myoglobin as a more sensitive early indicator of cardiac damage. More recently, the highly sensitive markers cardiac troponin subunits I and T have come to be preferred for their extraordinarily high specificity for identifying myocardial damage. These tests, along with other markers of skeletal muscle necrosis, provide a high degree of diagnostic accuracy. If performed in the emergency room an early and accurate diagnosis of myocardial damage offers great advantage to a suspected heart attack victim.
Diagnostic tests employing highly sensitive cardiac markers are described, for example, in U.S. Pat. Nos. 5,604,105 and 5,290,678. These procedures offer the rapidity of diagnosing myocardial infarction in the emergency room setting and offer significant medical benefit for patients.
Though numerous diagnostic assays for troponin subunits, principally I and T, exist, for example the troponin I Stratus(R) test from Dade International, Inc., the Opus(R) test from Behring, and the Access(R) test from Sanofi, there is no troponin subunit or complex preparation of sufficient quality and stability to employ as a standard for calibration of assays nor to use as a universally acceptable consensus standard across the industry. In order to maintain the conformational structure of troponin I, it must be complexed with troponin C. Troponin preparations from one source and for one particular assay format may not be useable in another, thus it is not currently possible to use the same standard across tests from different manufacturers. The lack of a industry-accepted standard limits the establishment of industry guidelines for test criteria as well as comparison of results throughout the world to help establish normal and abnormal value ranges.
Numerous troponin preparations from both natural and recombinant sources have been proposed. Previously described methods for the purification of troponin subunits from cardiac tissue (Tsukui et al., 1973, J. Biochem., v. 73, pp. 1119-1121; Cummins et al., 1978, Biochem. J., v. 171, pp. 251-259; Syska et al., 1974, FEBS Letts., v. 40, pp. 253-257) yielded preparations which were unstable and subject to considerable degradation on storage. A troponin complex prepared from cardiac tissue or from the combination of isolated troponin subunits prepared from cardiac tissue has been described (EP 0 743 522 A1). DE4405249 (U.S. Pat. No. 5,583,200) describes a stabilized troponin I or T also containing troponin C, with stability of several days in the cold. Use of a combination of four protease inhibitors in U.S. Pat. No. 5,560,937 overcame the degradation and stability issues inherent in tissue-derived troponin I, but did not obviate the need for human heart tissue. Unfortunately, human tissue is a potential source of infection, including HIV and hepatitis, to workers during troponin purification, and human heart tissue is of increasing scarcity especially with the successful use of early diagnostic tests for heart attack responsible for decreasing mortality from this disease. Troponin standards prepared by recombinant means offer a less costly and less hazardous alternative to that from heart tissue, but may still suffer from a lack of stability incompatible with industry demands.
Recombinant troponin subunits and complexes have been described. Armour et al. (1993, Gene, v. 131. pp. 287-292) cloned human cardiac troponin I in a bacterial system and expressed the gene product both as a beta-gal fusion product, and as an unfused product. Al-Hillawi et al. (1994, Eur. J. Biochem., v. 225, pp. 1195-1201) expressed human cardiac troponin I and troponin C in E. coli, using two codon changes in the cDNA of the former to overcome difficulties in expressing the human product in bacteria. Malnic and Reinach (1994, Eur. J. Biochem., v. 222, pp. A9-54) produced a recombinant complex in vivo by cloning all three chicken skeletal muscle troponin subunits into an expression plasmid. The troponin complex formed within the bacterium. Fujita-Becker et al. (1993, J. Biochem., v. 114, pp. 438-444) described the reconstitution of rabbit skeletal troponin complex from recombinant subunits expressed in E. coli. None of these recombinant products has been demonstrated to have adequate stability for use as a diagnostic test standard or calibrator. A recombinant human cardiac troponin complex formed in vitro from recombinant human cardiac troponin I, recombinant human cardiac troponin T, and recombinant human cardiac troponin C has not been described previously, nor has a complex formed of recombinant human cardiac troponin I and recombinant human cardiac troponin C.
Thus, there is a need for troponin complexes which meet the requirements of a stable material of safe origin and economical preparation that may be used as controls or calibrators across troponin assays. It has now been discovered that human cardiac troponin complexes prepared from recombinant subunits offer, among other advantages, superior stability and utility among troponin assays to be suitable for use as a universal standard.